Accurate Prediction of Inhibitor Binding to HIV-1 Protease Using CANDOCK

The human immunodeficiency virus 1 (HIV-1) protease is an important target for treating HIV infection. Our goal was to benchmark a novel molecular docking protocol and determine its effectiveness as a therapeutic repurposing tool by predicting inhibitor potency to this target. To accomplish this, we predicted the relative binding scores of various inhibitors of the protease using CANDOCK, a hierarchical fragment-based docking protocol with a knowledge-based scoring function. We first used a set of 30 HIV-1 protease complexes as an initial benchmark to optimize the parameters for CANDOCK. We then compared the results from CANDOCK to two other popular molecular docking protocols Autodock Vina and Smina. Our results showed that CANDOCK is superior to both of these protocols in terms of correlating predicted binding scores to experimental binding affinities with a Pearson coefficient of 0.62 compared to 0.48 and 0.49 for Vina and Smina, respectively. We further leveraged the Database of Useful Decoys: Enhanced (DUD-E) HIV protease set to ascertain the effectiveness of each protocol in discriminating active versus decoy ligands for proteases. CANDOCK again displayed better efficacy over the other commonly used molecular docking protocols with area under the receiver operating characteristic curve (AUROC) of 0.94 compared to 0.71 and 0.74 for Vina and Smina. These findings support the utility of CANDOCK to help discover novel therapeutics that effectively inhibit HIV-1 and possibly other retroviral proteases.

B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

ART duration and immunometabolic state determine efficacy of DC-based treatment restoring functional HIV- specific CD8+ T cells in Chronic PLWH.

Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated in detail. Here, we studied the association of ART duration with memory subsets, exhaustion and metabolic profiles of CD8+ T cells from PLWH and improvement of polyfunctional and effector HIV-1 specific responses after stimulation with Gag-adjuvant-primed DC. HIV-1-specific CD8+ T cell responses from a larger proportion PLWH on ART for more than 10 years (LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, CD8+ T cells from PLWH on ART for less than a decade (ST-ARTp) were less responsive to DC treatment and functional improvement was limited in this group. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolytic induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+PD1- cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines.

Raltegravir 1200 mg once daily as maintenance therapy in virologically suppressed HIV-1 infected adults: QDISS open-label trial

Background Raltegravir (RAL) has favorable tolerability and safety profile, with few and manageable drug interactions. The use of RAL 1200 mg once daily (qd) for first-line therapy is well established. We assessed efficacy and safety of RAL 1200 mg qd, as part of triple combined antiretroviral therapy (cART), for maintenance strategy. Methods The QDISS trial (NCT03195452) was a 48-week multicenter, single-arm, open-label study designed to evaluate the ability of 2 NRTIs + RAL 1200 mg qd to maintain virological suppression in HIV-1 infected subjects on a stable cART with 2 NRTIs and a third agent for at least 6 months. The primary endpoint was the proportion of participants with HIV-1 RNA < 50 copies/mL at week 24, by the FDA snapshot algorithm. Results Of 100 participants 91% maintained viral suppression (95% CI: 83.6–95.8) at week 24 and 89% (81.2–94.4) at week 48. At week 24, there was one virological failure, without emergence of resistance-associated mutation and 10 participants had discontinued, 4 because of adverse events (AEs). Over 48 weeks, 7 AEs of grade 3–4 were reported, one possibly study-drug related (spontaneous abortion). BMI remained stable regardless of previous therapy or baseline BMI category. Over 48 weeks, total cholesterol (p = 0.023) and LDL-cholesterol (p = 0.009) decreased, lifestyle and ease subscale significantly improved (p = 0.04). The quality of life and Patients Reported Outcomes (PROs) also improved at W12 (p = 0.007). Conclusion RAL 1200 mg qd as part of a maintenance triple therapy showed a high efficacy in virologically suppressed HIV-1 infected subjects, with good safety profile and improved lipid profile and patient reported outcomes. Trial registration: Clinical trials.gov NCT03195452 and EudraCT 2016-003702-13.

Off-label use of combined antiretroviral therapy, analysis of data collected by the Italian Register for HIV-1 infection in paediatrics in a large cohort of children

Background Early start of highly active antiretroviral therapy (HAART) in perinatally HIV-1 infected children is the optimal strategy to prevent immunological and clinical deterioration. To date, according to EMA, only 35% of antiretroviral drugs are licenced in children < 2 years of age and 60% in those aged 2–12 years, due to the lack of adequate paediatric clinical studies on pharmacokinetics, pharmacodynamics and drug safety in children. Methods An observational retrospective study investigating the rate and the outcomes of off-label prescription of HAART was conducted on 225 perinatally HIV-1 infected children enrolled in the Italian Register for HIV Infection in Children and followed-up from 2001 to 2018. Results 22.2% (50/225) of included children were receiving an off-label HAART regimen at last check. Only 26% (13/50) of off-label children had an undetectable viral load (VL) before the commencing of the regimen and the 52.0% (26/50) had a CD4 + T lymphocyte percentage > 25%. At last check, during the off label regimen, the 80% (40/50) of patients had an undetectable VL, and 90% (45/50) of them displayed CD4 + T lymphocyte percentage > 25%. The most widely used off-label drugs were: dolutegravir/abacavir/lamivudine (16%; 8/50), emtricitbine/tenofovir disoproxil (22%; 11/50), lopinavir/ritonavir (20%; 10/50) and elvitegravir/cobicistat/emtricitabine/ tenofovir alafenamide (10%; 10/50). At logistic regression analysis, detectable VL before starting the current HAART regimen was a risk factor for receiving an off-label therapy (OR: 2.41; 95% CI 1.13–5.19; p = 0.024). Moreover, children < 2 years of age were at increased risk for receiving off-label HAART with respect to older children (OR: 3.24; 95% CI 1063–7.3; p = 0.001). Even if our safety data regarding off-label regimens where poor, no adverse event was reported. Conclusion The prescription of an off-label HAART regimen in perinatally HIV-1 infected children was common, in particular in children with detectable VL despite previous HAART and in younger children, especially those receiving their first regimen. Our data suggest similar proportions of virological and immunological successes at last check among children receiving off-label or on-label HAART. Larger studies are needed to better clarify efficacy and safety of off-label HAART regimens in children, in order to allow the enlargement of on-label prescription in children.

Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda

Background Our understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia. Results In this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4+ T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4+ counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001). Conclusions The smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

HIV-1–Specific Immunodominant T-Cell Responses Drive the Dynamics of HIV-1 Recombination Following Superinfection

A series of HIV-1 CRF01_AE/CRF07_BC recombinants were previously found to have emerged gradually in a superinfected patient (patient LNA819). However, the extent to which T-cell responses influenced the development of these recombinants after superinfection is unclear. In this study, we undertook a recombination structure analysis of the gag, pol, and nef genes from longitudinal samples of patient LNA819. A total of 9 pol and 5 nef CRF01_AE/CRF07_BC recombinants were detected. The quasispecies makeup and the composition of the pol and nef gene recombinants changed continuously, suggestive of continuous evolution in vivo. T-cell responses targeting peptides of the primary strain and the recombination regions were screened. The results showed that Pol-LY10, Pol-RY9, and Nef-GL9 were the immunodominant epitopes. Pol-LY10 overlapped with the recombination breakpoints in multiple recombinants. For the LY10 epitope, escape from T-cell responses was mediated by both recombination with a CRF07_BC insertion carrying the T467E/T472V variants and T467N/T472V mutations originating in the CRF01_AE strain. In pol recombinants R8 and R9, the recombination breakpoints were located ~23 amino acids upstream of the RY9 epitope. The appearance of new recombination breakpoints harboring a CRF07_BC insertion carrying a R984K variant was associated with escape from RY9-specific T-cell responses. Although the Nef-GL9 epitope was located either within or 10~11 amino acids downstream of the recombination breakpoints, no variant of this epitope was observed in the nef recombinants. Instead, a F85V mutation originating in the CRF01_AE strain was the main immune escape mechanism. Understanding the cellular immune pressure on recombination is critical for monitoring the new circulating recombinant forms of HIV and designing epitope-based vaccines. Vaccines targeting antigens that are less likely to escape immune pressure by recombination and/or mutation are likely to be of benefit to patients with HIV-1.

HIV-1 and HTLV-1 Transmission Modes: Mechanisms and Importance for Virus Spread

So far, only two retroviruses, human immunodeficiency virus (HIV) (type 1 and 2) and human T-cell lymphotropic virus type 1 (HTLV-1), have been recognized as pathogenic for humans. Both viruses mainly infect CD4+ T lymphocytes. HIV replication induces the apoptosis of CD4 lymphocytes, leading to the development of acquired immunodeficiency syndrome (AIDS). After a long clinical latency period, HTLV-1 can transform lymphocytes, with subsequent uncontrolled proliferation and the manifestation of a disease called adult T-cell leukemia (ATLL). Certain infected patients develop neurological autoimmune disorder called HTLV-1-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP). Both viruses are transmitted between individuals via blood transfusion, tissue/organ transplantation, breastfeeding, and sexual intercourse. Within the host, these viruses can spread utilizing either cell-free or cell-to-cell modes of transmission. In this review, we discuss the mechanisms and importance of each mode of transmission for the biology of HIV-1 and HTLV-1.

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.